1. Field of Invention
This invention relates to a protein-free medium for the growth of hybridomas in suspension and spinner cultures.
2. Information Disclosure Statement
Hybridoma growth media typically contain serum. See Koprowski, U.S. Pat. No. 4,196,265; Wands, U.S. Pat. No. 4,271,145; Galfre, U.S. Pat. No. 4,350,683. However, Geltosky, U.S. Pat. No. 4,521,510 cultivated newly fused cells in a undisclosed serum-free medium.
Cleveland, et al., J. Immunol. Meth. 56:221 (1983) describes a protein-free medium for hybridoma cultivation. His basal medium was Ham's Fl2 and IMDM in 1:1 ratio, supplemented with NaHCO.sub.3, streptomycin, alphathioglycerol, and progesterone. For cultivation, it was supplemented with two trace element mixtures, providing Cu, Mn, Si, Mo, V, Ni, Sn, Zn, Se, Al, Ag, Ba, K, Cd, Co, Cr, Na, Fe, Ge, Rb and Zr. The medium did not contain any organo-iron compound, any selenium salt, or EDTA. The cells were gradually adapted to the protein-free medium. The tolerance of cells for protein-free conditions, and for rapid changes to protein-free conditions, varied from cell line to cell line. Significantly, all of Cleveland's successes were with cells grown as attached monolayers. Even in media supplemented with insulin and transferring, Cleveland was unable to keep hybridomas alive in stationary suspension culture.
Lymphocytes were cultured by Shive, PNAS 83:9 (1986). Shive requires the toxic HEPES buffer and lacks several amino acids (alanine, asparagine, aspartic acid, cystine, glutamic acid and proline), sodium nitroprusside, ions other than Ca, Fe, K, Mg, and Na, the preferred lipophilic components, and other preferred components. Shive, U.S. Pat. No. 4,499,064 emphasizes the inclusion of HEPES buffer and phytohemagglutinin (a glycoprotein). There is some contradiction between the article and the patent as to riboflavin level. While Shive's medium included EDTA, it did not provide SNP or a selenium compound.
Basset, et al. in HORMONALLY DEFINED MEDIA: A TOOL IN CELL BIOLOGY, 219 (1983) grew L1210 leukemic cells at a seed density of 4.times.10.sup.4 cells/ml in RPMI 1640 in the presence of either 10% FCS or of the guanylate cyclase activators hemin or sodium nitroprusside. Bassett's medium did not contain EDTA or a selenium salt. L1210 leukemic cells have different growth requirements than hybridoma cells, as is shown by my observation (See Detailed Description) that Bassett's medium did not support the growth of hybridoma cells HB60 or 1410 at any cell density tested.
Basset, et al. Cancer Res., 46:1644-47 (1986) showed that L1210 cells could grow in the absence of serum and transferring when ferrous sulfate or ferric citrate was provided in a concentration of 1-100 .mu.M. They also studied the effect of desferrioxamine, an iron chelator, on L1210 cells established in transferring-supplemented serum-free medium, finding that prolonged exposure to desferrioxamine had a detrimental effect on cell viability. The addition of ferric citrate or ferrous sulfate to the desferrioxamine-treated cells reinitiated DNA synthesis, but potassium ferricyanide was much less effective. The reinitiation of DNA synthesis and cell growth by ferric citrate was reversed by the antioxidant propyl gallate. Thus, Basset taught against the combination of an iron chelator such as EDTA or an antioxidant known to inhibit lipid peroxidation induced by iron, with an iron transport compound like SNP.
Several references teach use of inorganic iron salts in defined media containing protein. Perez-Infante and Mather, Exper. Cell Res., 142:325-32 (1982) cultivated anchorage-dependent mouse testicular cell lines in a medium consisting of Ham's 1212, insulin, EGF. Hepes, gentamicin and FeSO.sub.4. Taetle, et al., J. Clin. Invest., 75:1061-67 (Mar. 1985) describes growth of HL-60 and KG-1 leukemic cells in a medium consistion of RPMI-1640, insulin, ethanolamine, selenium and ferric nitrilacetate. Titeux, et al., J. Cellular Physiol., 121:251-56 (1984) propagated the erythroleukemia K562 and HEL cell lines in a medium made by combining RPMI-1640, albumin, insulin and ferric ammonium citrate. Titeux discouraged use of iron chelators since they completely inhibited cell growth. Phillips and Cristofalo, Exper. Cell Res., 134:297-302 (1981) grew anchorage-dependent WI-38 (normal human diploid fibroblast-like) cells in MCDB-104 supplemented with EGF, insulin, dexamethasone and ferrous sulfate. Amauric, et al., In Vitro, 20:543-548 (1985) cultivated anchorage dependent HT 29 cells in 1:1 Ham's F12:DMEM supplemented with Hepes, selenium, EGF and ferrous sulfate (or ferrous chloride).
Terada U.S. Pat. No. 4,134,793 discloses the use of sodium nitroprusside in the indophenol test for urea. Terada is pertinent only to the extent that he shows that sodium nitroprusside is a known compound. Terada does not, however, use it to support the growth of cells. Terada disrupts bacterial cells before adding SNP to the lysate. It would not be obvious from Terada's work to use SNP in a culture medium.
Kellner U.S. Pat. No. 3,929,582 discloses the use of ferric ammonium citrate in a bacterial cell culture. However, the nutritional requirements of bacterial cells are different from those of hybridoma cells, hence, its use in cultivating hybridomas would not be obvious from Kellner's teaching.
Yoshimoto, U.S. Pat. No. 4,172,572 cultured hybridomas in spinner culture, however, he used an RPMI-1640 medium containing 10% FCS.
Jakoby, et al., Methods in Enzymology 58:62-71 (1979) summarizes the composition of 27 different culture media. Of all of these media, only one, Higuchi's, is said to contain EDTA. However, the same table reveals that it lacks selenium. Only the MCDB family of media (301, 105, 202, 501, 401 and 411) were declared to contain selenium, but, of course, they do not include EDTA. None of Jakoby's 27 media contain sodium nitroprusside.
Torney, U.S. Pat. Nos. 3,887,430 and 4,055,466 describe a chemically defined protein-free medium comprising sugar, amino acids, mineral salts; vitamins, a water-soluble lipid source (preferably a polysorbate, but may be sodium oleate or stearate) and a basic anion exchange resin. This medium was used for chick embryo tissue culture and to support the anchorage-dependent MDBK cell line. The resin may hinder subsequent purification steps.
Serum-free media are well known. That of Stemerman, U.S. Pat. No. 4,443,546 contained EGF, Cohn fraction IV, thrombin, insulin, transferring and/or ECGF.
U.S. Pat. No. Re. 30,985 describes a serum-free medium which contains insulin and therefore is not protein free. It is noteworthy that it presents a riboflavin level of 0.1 mg/L, and that it contains lipophilic components (linoleic, linolenic, or arachidonic acid).
The culture medium of Yamane, U.S. Pat. No. 4,533,637 contains insulin and transferring. Riboflavin is present at 0.4 mg/L. The lipophile provided is linoleic, oleic, linolenic, palmitic or stearic acid. Cyclodextrin is a preferred component.
Baker, U.S. Pat. No. 4,560,655 emphasizes use of a phosphatidylcholine as a lipid source. The other major components of his medium are fetuin and transferrin. Riboflavin is provided at a concentration of 0.4 mg/L.
Fabricius, U.S. Pat. No. 4,406,830's serum-free medium contained only one protein, his novel 90 Kd glycoprotein.
The serum-free medium of Tomei, U.S. Pat. No. 4,049,494 contains 0.1 mg/L of riboflavin. There is no reference to use of proteins.
Yabe, In Vitro Cell. & Dev. Biol., 23:815 (Dec. 1987) favors use of weak iron chelators as transferrin substitutes in serum-free growth of hybridomas. His medium contained selenium but not SNP. Use of EDTA wa reported to have inhibited cell growth, leading to cell death.
No admission is made that any of the foregoing constitute prior art. All references are incorporated by reference to the extent pertinent.